PREECLAMPSIA (V GAROVIC, SECTION EDITOR)

Preeclampsia and Extracellular Vesicles

Sarwat I. Gilani1,2 & Tracey L. Weissgerber1 & Vesna D. Garovic1 &

Muthuvel Jayachandran2,3

Published online: 2 September 2016# The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract Preeclampsia is a hypertensive pregnancy disor-der characterized by development of hypertension and pro-teinuria after 20 weeks of gestation that remains a leadingcause of maternal and neonatal morbidity and mortality.While preeclampsia is believed to result from complex in-teractions between maternal and placental factors, the prox-imate pathophysiology of this syndrome remains elusive.Cell-to-cell communication is a critical signaling mecha-nism for feto-placental development in normal pregnancies.One mechanism of cellular communication relates to acti-vated cell-derived sealed membrane vesicles called extra-cellular vesicles (EVs). The concentrations and contents ofEVs in biological fluids depend upon their cells of originand the stimuli which trigger their production. Research onEVs in preeclampsia has focused on EVs derived from thematernal vasculature (endothelium, vascular smooth mus-cle) and blood (erythrocytes, leukocytes, and platelets), as

well as placental syncytiotrophoblasts. Changes in the con-centrations and contents of these EVs may contribute to thepathophysiology of preeclampsia by accentuating the pro-inflammatory and pro-coagulatory states of pregnancy.This review focuses on possible interactions amongplacental- and maternal-derived EVs and their contents inthe initiation and progression of the pathogenesis of pre-eclampsia. Understanding the contributions of EVs in thepathogenesis of preeclampsia may facilitate their use asdiagnostic and prognostic biomarkers.

Keywords Hypertensive pregnancy disorder .

Microvesicles . Exosomes . Cell-cell communication .

Vesicles

Introduction

Preeclampsia is characterized by new-onset hypertension (sys-tolic blood pressure ≥140 mmHg/diastolic blood pressure≥90 mmHg), with either proteinuria (≥300 mg/24 h) and/ororgan dysfunction after 20 weeks of gestation [1]. The under-lying cellular and molecular mechanisms that trigger pre-eclampsia and facilitate its progression are not well understood.Consequently, there are no established early diagnostic tests oreffective targeted pharmacological treatments for preeclampsia.The only treatment option is delivery. With a global prevalencerate of 2.7–8.2% of pregnancies, preeclampsia remains a majorchallenge in patient management for physicians [2–4].

It is recognized increasingly that preeclampsia is a hetero-geneous disease, caused by several distinct underlying mech-anisms that may result in different clinical phenotypes [5••].This is reflected in current clinical practice, as it is common todivide preeclampsia into early (<34 weeks of gestation) andlate (>34 weeks of gestation) preeclampsia based on the

This article is part of the Topical Collection on Preeclampsia

* Muthuvel Jayachandranjaya.m@mayo.edu

Sarwat I. Gilanigilani.sarwat@mayo.edu

Tracey L. Weissgerberweissgerber.tracey@mayo.edu

Vesna D. Garovicgarovic.vesna@mayo.edu

1 Department of Internal Medicine, Division of Nephrology andHypertension, Mayo Clinic, Rochester, MN 55905, USA

2 Department of Surgery, Mayo Clinic, 200 First Street SW,Rochester, MN 55905, USA

3 Department of Physiology and Biomedical Engineering, MayoClinic, Rochester, MN 55905, USA

Curr Hypertens Rep (2016) 18: 68DOI 10.1007/s11906-016-0678-x

timing of the onset of symptoms. Similarly, preeclampsiamay be classified as mild or severe depending on the se-verity of symptoms, including blood pressure (mild, <160/110 mmHg; severe, ≥160/110 mmHg), and the presence orabsence of organ dysfunction (kidney failure, liver rup-ture, stroke, and seizure). Studies investigating the etiolo-gies of preeclampsia have hypothesized that this syndromemay have placental and maternal forms [6]. This approachtakes into account the underlying mechanisms. It has beenproposed that defects in remodeling of the maternal spiralarteries that supply the placenta ultimately lead to placen-tal ischemia [7••], ischemia reperfusion injury [8], or highvelocity blood flow injury in the intervillous space [9••] inplacental preeclampsia. This triggers the release of one ormore placental factors that cause systemic endothelial dys-function in the maternal circulation. Alternatively, mater-nal preeclampsia may arise in the setting of vascular dys-function, oxidative stress, and metabolic abnormalities,such as hypertension, obesity, or diabetes that predate orare exacerbated by pregnancy (in the text that follows, wewill refer to these conditions as preeclampsia risk factors).Endothelial dysfunction worsens with advancing gestationas the mother is unable to adapt to the physiological stressof pregnancy (Fig. 1). Placental preeclampsia is common-ly viewed as early or severe, while maternal preeclampsiais sometimes characterized as late or mild. Although thedichotomous view of preeclampsia is overly simplistic, therelative contributions of maternal vs. placental factorslikely differ among individual women, ultimately resultingin a diverse spectrum of clinical presentations. Irrespectiveof the predominant underlying mechanism, the interac-tions among maternal and placental pathophysiologicalfactors may lead to a vicious cycle of maternal inflamma-tion, vascular dysfunction, and the activation of pro-

coagulation pathways that ultimately cause the symptomsand signs of preeclampsia.

Based on the complex nature of the origin of pre-eclampsia, we hypothesize that placental and maternalcells cross-talk, mediated by extracellular vesicles (EVs),contributes to the initiation and progression of preeclamp-sia in women, both with and without known pre-existingrisk factors (Fig. 1). In women for whom EVs derivedfrom the placenta are the major contributors, we proposethat the symptoms of preeclampsia may appear earlier ingestation. If EVs derived from maternal cells are the majorcontributors, the symptoms may appear later in gestation.Two distinct types of EVs (exosomes and microvesicles)are released by almost all activated cells or cells involvedin pathophysiological processes [10–13]. Exosomes andmicrovesicles differ in size and their modes of formation.Exosomes are smaller than microvesicles (30–120 nm vs.40–1000 nm) [12], and are formed by the endocytosis ofmultivesicular bodies and are released from cells by exo-cytosis. In contrast, microvesicles (MVs) are membrane-bound vesicles that are shed from the plasma membrane[12]. Despite these differences, the size ranges for thesetwo distinct classes of EVs overlap in some extent, andthere are currently no established methods available todistinguish them purely on basis of size. Surface-specificEV markers that can be used to differentiate microvesiclesand exosomes have not yet been identified. We have there-fore used the term EVs, as previously suggested by thescientific community [14], to refer to exosomes andmicrovesicles in this review.

As the role of EVs in the pathophysiology of preeclampsiais an emerging field, the literature contains conflicting data.This review focuses on the most consistent findings, whileproviding an overview of areas with disparate findings.

Fig. 1 Role of EVs inpathogenesis of preeclampsia.Maternal risk factors andplacental abnormalities causesystemic maternal cell activationresulting in release of EVs.Endothelial-, leukocyte-, andplatelet-derived EVs give rise tovascular dysfunction, immunemodulation, and increasedthrombotic propensity. Theseprocesses collectively contributeto progression of pathogenesis ofpreeclampsia

68 Page 2 of 11 Curr Hypertens Rep (2016) 18: 68

Maternal Cell-Derived EVs Before Pregnancy

Changes in circulating EVs offer a unique opportunity to ex-amine how preeclampsia risk factors affect the functions of theparent cells and tissues prior to and during pregnancy. Riskfactors for preeclampsia include obesity, pre-gestational dia-betes mellitus, hypertension, and systemic lupus erythemato-sus (Table 1). These risk factors could alter the functioning ofdifferent types of maternal cells prior to pregnancy, as dem-onstrated by changes in the concentrations and bioactive mo-lecular contents of the circulating EVs. Studies examiningEVs in non-pregnant women suggest that risk factors for pre-eclampsia are associated with changes in EVs derived fromvascular endothelial cells, leukocytes, and platelets [17–26](Table 2). As shown in Table 2, compared to non-pregnantwomen without preeclampsia risk factors, non-pregnant wom-en with these risk factors are reported to have increasedendothelial-derived EVs [17–19, 22, 24, 25, 27]. Similarly,non-pregnant women with these risk factors are reported tohave increased platelet-derived EVs [17, 20–22, 26]; however,they have either increased or decreased concentrations ofleukocyte-derived EVs [20, 23–25].

Studies have shown that the effects of preeclampsia-associated risk factors are similar to the effects of preeclamp-sia on endothelial-derived EVs. Women with bothpreeclampsia-associated risk factors and preeclampsia are re-ported to have increased concentrations of endothelial-derivedEVs when compared to either non-pregnant women withoutthese risk factors and/or normotensive pregnant women, re-spectively [17–19, 22, 24, 25, 27–29]. However, some studiesreport no change in endothelial-derived EV concentrations inwomen with preeclampsia compared to normotensive preg-nant women [30–32]. Concentrations of leukocyte-derivedEV (LEV) in non-pregnant women with preeclampsia-associated risk factors are reported to be increased, decreased,or not changed, when compared to non-pregnant womenwith-out these risk factors [20, 23–25]. Whereas in women withpreeclampsia, LEV concentrations are reported to be

increased compared to normotensive pregnant women [28,33]. The effects of preeclampsia-associated risk factors arereported to be opposite to the effects of preeclampsia onplatelet-derived EV (PEV). Increased concentrations of PEVare present in women with preeclampsia-associated maternalrisk factors compared to non-pregnant women without theserisk factors. However, PEV concentrations are reported to bedecreased in women with preeclampsia compared to normo-tensive pregnant women [17, 20–22, 26].

Use of low-dose aspirin is recommended for women withpreeclampsia-associated risk factors to decrease the morbidityand mortality associated with preeclampsia [34]. The benefi-cial effects of aspirin in those with preeclampsia-associatedrisk factors may, at least in part, be explained by the effectof aspirin on platelet activity. By inhibiting thromboxane A2

synthesis, aspirin decreases platelet activation and, in turn,likely affects the production of PEV. Understanding the effectof aspirin on PEV production and content in women withpreeclampsia-associated risk factors may delineate the mech-anistic pathways by which PEV contribute to the pathogenesisof preeclampsia.

Placenta-Derived EVs

The placenta plays a critical role in the pathophysiology ofpreeclampsia [35]. Placental trophoblasts are involved in spi-ral artery remodeling and differentiate into extravillous tro-phoblasts and villous trophoblasts. The villous trophoblastsfuse to form syncytiotrophoblasts. The extravillous tropho-blasts invade the distal portions of the spiral arteries,displacing maternal vascular endothelial and smooth musclecells [36, 37]. This process transforms the distal portions ofthe spiral arteries from narrow vessels into wide, flaccid con-duits [38, 39•]. The uterine oxygen gradient in early pregnan-cy favors extravillous trophoblast invasion of the uterine spiralarteries and spiral artery remodeling [40]. It is speculated thatplacental trophoblast-derived EVs (40–300 nm) [41] may also

Table 1 Preeclampsia-associatedmaternal risk factors Pre-pregnancy maternal characteristics Relative risk Reference

Obesity 2.47 Duckitt et al. [15]

Pre-gestational diabetes mellitus 3.56 Duckitt et al. [15]

Hypertension 1.38–2.37 Duckitt et al. [15]

Autoimmune diseases

• Systemic lupus erythematosus –

• Antiphospholipid syndrome 9.72 Duckitt et al. [15]

Sickle cell anemia 2.43 Oteng-Ntim et al. [16]

Nulliparity 2.91 Duckitt et al. [15]

Preeclampsia in prior pregnancy 7.19 Duckitt et al. [15]

Family history of preeclampsia 2.90 Duckitt et al. [15]

Curr Hypertens Rep (2016) 18: 68 Page 3 of 11 68

have a role in spiral artery remodeling [42]. Salomon et al.have shown that oxygen tension regulates the number andprotein content of exosomes released by the placenta, withgreater release of exosomes by placental trophoblasts underhypoxic conditions in vitro [43, 44]. Placental exosomes arereported to contain serine proteases and metalloproteases(MMP), including MMP-12 [44]. It has been hypothesizedthat MMP-12 secreted by trophoblasts may facilitate tropho-blast invasion by contributing to the remodeling of the extra-cellular matrix in the vascular wall [45].

Abnormal placentation in women with preeclampsiamay increase circulating concentrations of placental-derived EVs. Studies have shown higher concentrationsof syncytiotrophoblast- derived EVs [28, 46, 47•, 48],with altered lipid and protein content [49, 50], in womenwith early-onset or severe preeclampsia compared to nor-motensive pregnant women. In addition, studies suggestthat the syncytiotrophoblast apoptosis rate is elevated inpreeclampsia (5–6 %) when compared to normal pregnan-cy (2–3 %) [51]. In accordance with this finding, highercirculating concentrations of syncytiotrophoblast-derivedEVs have been reported in preeclamptic women comparedto normotensive pregnant women [41, 46, 47•] .Furthermore, women with early-onset preeclampsia seemto have higher syncytiotrophoblast-derived EVs concen-trations than women with late-onset preeclampsia [28,48, 52•]. Further studies are needed to identify the exactcellular or molecular pathways that stimulate productionof placental-derived EVs, which may contribute to thedevelopment of preeclampsia.

Effect of Maternal EVs on Productionof Placental-Derived EVs

Dynamic interactions between maternal and fetal factorsare constantly occurring at the maternal-fetal interface.These interactions contribute to the regulation of tropho-blast phenotype and endovascular invasion. Chemokinesand their receptors (CXCR4, CXCR7, CXCL12) promotecell survival and proliferation and inhibit apoptosis [53].Lu et al. [53] have shown decreases in the expressions ofCXCR4, CXCR7, and CXCL12 molecules in trophoblastcells obtained from the placentas of preeclamptic women.The causes of these decreases are not known. One possiblemechanism may be that circulating maternal factors down-regulate chemokine receptors at the post-transcriptionallevel. Alternatively, downregulation of molecules or up-regulation of molecular inhibitors at the transcriptionallevel can also occur in trophoblasts, as demonstrated byZhou et al. [54]. This study also observed an upregulationof the angiogenesis inhibitor SEMA3B in trophoblasts ob-tained from women with preeclampsia. SEMA3B inhibitstrophoblast invasion of vessels by promoting trophoblastapoptosis. It additionally showed that the increased levelsof SEMA3B from preeclamptic trophoblasts returned tocontrol levels after 48 h in a culture system. Based on thisfinding, the authors proposed that factors in the maternalmilieu cause reversible upregulation of SEMA3B in tro-phoblasts in preeclampsia [54]. The role of maternal cell-derived EVs in regulating trophoblast gene expression inpreeclampsia remains to be determined. Holder et al. [55]

Table 2 EVs in non-pregnantwomen with preeclampsia-associated risk factors comparedto non-pregnant women withoutpreeclampsia-associated riskfactors

Risk factor Extracellular vesicles Results Reference

Obesity Endothelial-derived ↑ Stephanian et al. [17]

Platelet-derived ↑ Stephanian et al. [17]

Diabetes mellitus Endothelium-derived ↑ Sabatier et al. [18]

↑ Tramontano et al. [19]

Leukocyte-derived No difference Zhang et al. [20]

Platelet-derived ↑ Zhang et al. [20]

↑ Strano et al. [21]

Hypertension Endothelium-derived ↑ Preston et al. [22]

Platelet-derived ↑ Preston et al. [22]

Systemic lupus erythematosus Leukocyte-derived ↑ Lacroix et al. [27]

↓ Neilson et al. [24]

Endothelium-derived ↑ Lacroix et al. [27]

No difference Neilson et al. [24]

Platelet-derived ↑ Stephanian et al. [17]

Antiphospholipid syndrome Endothelium-derived ↑ Dignat-George et al. [25]

Leukocyte-derived ↑ Dignat-George et al. [25]

Sickle cell anemia Platelet-derived ↑ Wun et al. [26]

68 Page 4 of 11 Curr Hypertens Rep (2016) 18: 68

have shown that placental trophoblasts take up exosomesfrom maternal macrophages and alter the placental pro-duction of inflammatory cytokines. Given this evidence,we hypothesize that EVs derived from maternal cells havethe potential to alter trophoblast gene expression and func-tion. These changes may contribute to defective tropho-blast invasion and increased trophoblast apoptosis.

Placental debris is cleared by macrophages at thematernal - fe ta l in te r face [56] . I t i s known tha tsyncytiotrophoblast-derived vesicles affect the functionsof maternal cells, including platelets, leukocytes, erythro-cytes, and endothelial cells [57•, 58–62]. Therefore, it ispossible that EVs produced by syncytiotrophoblasts alsocontribute to regulating maternal macrophage activity atthe maternal-fetal interface. Exploring the effects ofsyncytiotrophoblast-derived EVs on maternal macro-phages will elucidate the mechanisms by which macro-phage activity is regulated at the maternal-fetal interface.The role of maternal EVs in the regulation of trophoblastturnover and immune activity at the maternal-fetal inter-face will provide valuable insight into the factors regulat-ing the dynamics of the maternal-fetal interface. This, inturn, will delineate the role that EVs of maternal andplacental origins and their interactions have in the initia-tion and progression of placental preeclampsia.

Maternal Cell-Derived EVs and Their Interactions

Platelet-Derived EVs Platelets are the largest source of EVsin blood in healthy non-pregnant women [63]. When com-pared to non-pregnant women, most studies report lowerplatelet-derived EV(PEV) concentrations in normotensivepregnant women [28, 64]. While most studies have observedfurther reductions in PEV in women with preeclampsia [28,31, 32, 65], a few studies have shown higher concentrations ofPEV, or no change [66, 67] (Table 3). It is plausible that dif-ferences in the reported concentrations of PEV among thestudies are reflective of different preeclampsia subtypes andtheir underlying mechanisms. Three different theories havebeen proposed to explain the lower concentrations of PEVwith preeclampsia. (1) Lower platelet counts in preeclampsiamay contribute to lower concentrations of PEV [65]. (2) Somestudies have hypothesized that the lower concentrations ofPEV may partly be due to increased trapping or participationof PEVs in thrombin generation and fibrin clot formation [28,64, 67, 72]. (3) It is postulated that lower PEVs may be due totheir association or binding with leukocytes [65].

Leukocyte-Derived EV Studies have shown that leukocytecounts and concentrations of leukocyte-derived EV (LEV) arehigher in normotensive pregnant women compared to non-

Table 3 Comparison of EVs between preeclampsia and normotensive pregnancy

Parameter Preeclampsia Normotensivepregnancy

Reference

Total EVs Increased/nochange/decreased

Present Tesse et al. [68]; Marques et al. [28];Mikhailova et al. [33]; VanWijk et al. [69];Holthe et al. [70]; Bretelle et al. [32];Lok et al. [31]

Syncytiotrophoblast-derivedEVs (early-onset PE)

Increased/nosignificant change

Present Knight et al. [46]; Germain et al. [47•];Goswami et al. [48]; Lok et al. [31]

Syncytiotrophoblast-derivedEVs (late-onset PE)/severe

No change/no change Present Goswami et al. [48]; [28]

Endothelial cell-derived EVs Increased/no change Present Marques et al. [28]; Gonzalez et al. [29];VanWijk et al. [69]; Bretelle et al. [32];Lok et al. [31]

Platelet-derived EVs Decreased Present Marques et al. [28]; Bretelle et al.[32];Lok et al.[28, 31]; Lok et al. [65]

Leukocyte-derived EVs Increased Present Mikhailova et al. [33]; Marques et al. [28]

• Granulocyte-derived EVs Increased Present Lok et al. [71]; vanWijk et al. [69];Marques et al. [28]

• Monocyte-derived EVs Increased Present Lok et al. [31, 71]; Marques et al.[28]

• Lymphocyte-derived EVs Decreased Present Lok et al. [71]; Marques et al.[28]

• T cell-derived EVs Increased Negligible vanWijk et al.[69]

• T helper cell-derived EVs Increased Negligible Lok et al. [31]

Erythrocyte-derived EVs Increased Present Lok et al. [31]; Marques et al. [28];Dragovic et al. [64]

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pregnant women [28, 33, 64, 69, 71, 73]. The inflamma-tory state of normotensive pregnancy is further exacerbat-ed in preeclampsia, as preeclamptic women have evenhigher leukocyte counts and concentrations of LEV [64,71] (Table 3). The upregulation of granulocytes (neutro-phils)-and monocyte- and granulocyte-derived EVs-hasbeen suggested [28, 33, 71] to aid in the removal andregulation of syncytiotrophoblast-derived vesicles andplacental debris that are released into the maternal circu-lation. Pro-inflammatory EVs can be produced by endo-thelial cells in response to inflammatory stimuli [74].Therefore, we speculate that the pro-inflammatory statein preeclampsia may increase circulating concentrationsof endothelial-derived pro-inflammatory EVs. These pro-inflammatory EVs may contribute to the increases in theperipheral blood neutrophils and monocytes in preeclamp-sia, facilitating the immunomodulation and upregulationof phagocytosis.

The role of EVs in leukocyte activation has also been ex-plored. Studies have shown that syncytiotrophoblast-derivedvesicles from normal pregnancy and preeclampsia cause leu-kocyte activation in vitro [47•, 75] that is mediated by toll-likereceptors and nuclear factor (NF-Kβ) [76]. Peripheral bloodEVs have also been shown to alter monocyte phenotypein vitro [60]. Furthermore, it has been shown that activatedleukocytes produce inflammatory cytokines (IL-1, IL-8) andnuclear factor (NF-Kβ) that can stimulate EV production [77].The circulating EVs of maternal and placental origins mostlikely propagate inflammation in preeclampsia [37].

Red Blood Cells (Erythrocytes)-Derived EV While moststudies show an increase in the concentrations of red bloodcells (erythrocytes)-derived EV (REV) in normotensive preg-nant women and preeclamptic women compared to non-pregnant women [28, 31, 64], one study showed no difference[64]. Increased REV concentrations in pregnancy suggesterythrocyte activation, which may be due to increased oxygendemand in pregnancy or stimulation by circulating EVs.Alternatively, increased REV concentrations in preeclampsiamay be due to erythrocyte disruption and hemolysis [78],which may be associated with widespread thrombosis [28].Ten to twenty percent of women with preeclampsia develophemolysis, elevated liver enzymes, and low platelets (HELLP)syndrome, which is characterized by erythrocyte disruption[79]. Hemolysis, in addition, may result from an autoimmunereaction to trophoblast-derived vesicles deposition on erythro-cytes that may explain the increased concentrations of REV inpreeclampsia. Determination of REV concentrations and com-position in pregnant women with and without preeclampsiaand with HELLP syndrome may help to elucidate the mech-anisms by which preeclampsia progresses to HELLPsyndrome.

Vascular Endothelium and Smooth Muscle Cell-DerivedEVs The concentrations of endothelial-derived EV (EEV)are lower in normotensive pregnant women compared tonon-pregnant women, reflecting either decreased produc-tion of EEV in normotensive pregnancy or EEV bindingto circulating blood cells (platelets and leukocytes).Decreased EEV production is associated with decreasedperipheral vascular resistance in normotensive pregnancy[80, 81]. Estrogen and maternal fluid dynamics [82] havebeen postulated to decrease EEV production in normalpregnancy. As syncytiotrophoblast-derived vesicles carrybioactive molecules (e.g., mRNA or miRNA), it may bepossible that they regulate the endothelial functions asso-ciated with decreased peripheral vascular resistance.Petrozella et al. [83] and VanWijk et al. [69] have shownthat circulating EEV concentrations were higher in pre-eclamptic women compared to normotensive pregnantwomen (Table 3). This indicates activation of endothelialcells in preeclampsia and an association between in-creased concentrations of EEV and vascular dysfunction.It has been demonstrated that EEV have differing charac-teristics depending on signaling stimulus and associatedthrombotic and inflammatory processes or conditions[84]. In addition to negatively charged phospholipids,the surface of EEV display receptors (E-selectin, intercel-lular adhesion molecule-1, and vascular cellular adhesionmolecule-1) and markers expressed by endothelial cells.Determining the composition and surface expressions ofEEV in normotensive pregnant and preeclamptic womenmay elucidate the interactions that result in the exacerba-tion of inflammation and coagulation activation in pre-eclampsia. EEVs are implicated in the progression of in-flammatory vascular diseases [85]. EEV-mediated com-munication between endothelial cells and the target cells(leukocytes, platelets) is vital to the understanding of theexaggerated pro-inflammatory and pro-coagulation statesunderlying preeclampsia. Vascular smooth muscle cell-derived EVs (SMCEVs) have also been implicated inpathological processes resulting in vascular disease pro-gression [86]. The contributions of SMCEVs in the path-ophysiology of preeclampsia have not been investigated.Studies exploring the role of SMCEVs may delineate ad-ditional mechanistic vascular pathways contributing to theinitiation and progression of preeclampsia.

EVs and Coagulation in Preeclampsia

The pro-coagulation state of normotensive pregnancy is asso-ciated with a decrease in fibrinolytic activity caused by in-creased pro-coagulant factors and fibrinolytic inhibitors(e.g., plasminogen activator inhibitor-1, [PAI-1]). In addition,the phosphatidylserine present on the surfaces of placental and

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maternal cell-derived EVs contributes to the hypercoagu-lable state. Thrombin generation and prothrombin frag-ments increase in normotensive pregnancy [87]. This hy-percoagulable state is exaggerated in preeclampsia,resulting in widespread blood clot formation [88], withfibrin deposition in the maternal vasculature, organs, andthe placenta [89]. This may be due to the increased totalconcentrations of pro-coagulant surface positive EVs;however, certain studies have shown no change or evendecreases in total counts of MVs in preeclampsia.Alternatively, changes in the phenotype of EVs could alsocause increased coagulation. Certain pro-coagulatory mol-ecules (e.g., PAI-1) also have important roles in EEVgeneration [90, 91]. Studies exploring the link betweenpro-coagulant factors and EV generation in preeclampsiamay elucidate the mechanism that links endothelial dys-function with widespread coagulation. Depending uponthe severity of preeclampsia, widespread coagulation ac-tivation and clot formation results in ischemic damage inend organs, as well as widespread disseminated intravas-cular coagulation.

Tissue factor is a ubiquitous 47 kDa transmembrane proteinthat initiates the inflammatory coagulation pathway. It is pres-ent on cells, as well as on cell-derived EVs. It is constitutivelyexpressed in some cells (perivascular fibroblasts) and condi-tionally expressed in other cells in response to a variety ofstimuli, including activated monocytes, macrophages, andthe vascular endothelium [92]. The EVs released from activat-ed leukocytes and endothelial cells also express tissue factor[63]. Previous studies have demonstrated that tissue factor ispresent on the surface of syncytiotrophoblast-derived vesicles[93, 94]. Upregulation of tissue factor on syncytiotrophoblastsoccurs in preeclampsia [95], which is associated with the in-creased activity of tissue factor in preeclampsia [96]. Gardineret al. [96] have demonstrated higher tissue factor activity andthrombin generation associated with syncytiotrophoblast-derived vesicles from preeclamptic women compared to nor-motensive pregnant women. Preclinical studies have revealedimproved clinical outcomes following anticoagulant therapiesin animal models of preeclampsia [97]. Human studies thatexplore the use of anticoagulants suitable for preeclampticwomen are needed. Based on animal studies, anticoagulanttherapy has the potential to improve maternal and fetal out-comes in preeclamptic pregnancies.

Conclusions

EVs have a dynamic role in the communication amongmaternal vascular cells (the vascular endothelium, circu-lating leukocytes, and platelets) and the placenta, thuscontributing to the progression of normal pregnancy.Depending on pre-existing maternal conditions, any of

these vascular components during pregnancy may be ca-pable of initiating the cascade of events that result inpreeclampsia. In maternal conditions associated with theactivation of vascular endothelial cells and immune sys-tem modulation, EEVs can augment inflammation, coag-ulation, and endothelial dysfunction. Pre-pregnancy ma-ternal platelet activation can augment endothelial dysfunc-tion and inflammation via PEVs, facilitating the progres-sion to preeclampsia. In women without maternal riskfactors associated with preeclampsia, it is possible thatplacental trophoblast-derived EVs may contribute to thematernal milieu that favors progression to preeclampsia.

Future Directions The complex interactions of maternalcell-derived EVs and placental-derived EVs need to beexplored further to elucidate the mechanisms of initiationand progression of preeclampsia, with and without knownmaternal risk factors. In addition to quantitative alter-ations in EVs, characterizing the bioactive molecular con-tents (mRNA, miRNA, proteins, lipids, and metabolites)of these EVs based on their cellular origins and their in-teractions with target cells in the blood and vascular com-partments may help to identify underlying mechanismsthat contribute to the pathophysiology of preeclampsia.Furthermore, understanding the roles of the specific typesof EVs in the pathogenesis of preeclampsia may enablethe development of a panel of biomarkers that will help toidentify pregnant women at risk for developing pre-eclampsia. In addition, as maternal vascular, immune,and coagulation systems may have EV-mediated bidirec-tional communication, it may be possible to therapeutical-ly target one component of the maternal system, whichmay facilitate the other two systems to return to normalcyin preeclampsia. As most EVs in the healthy pregnantstate are derived from platelets, therapeutic interventionsaimed at stabilizing platelets can correct the exaggeratedpro-inflammatory and hypercoagulable state in pre-eclampsia that may also help to mitigate the vascular dys-function and immune flare.

Acknowledgments This study was supported by award numberP50-AG44170 (V.D.G. and M.J.) from the National Institute onAging; by the Building Interdisciplinary Careers in Women’sHealth award K12HD065987 (T.L.W.) from the Office ofWomen’s Health Research.

Compliance with Ethical Standards

Conflict of Interest Drs. Gilani, Weissgerber, Garovic, andJayachandran declare no conflicts of interest relevant to this manuscript.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects performed by anyof the authors.

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Open Access This article is distributed under the terms of the CreativeCommons At t r ibut ion 4 .0 In te rna t ional License (h t tp : / /creativecommons.org/licenses/by/4.0/), which permits unrestricted use,distribution, and reproduction in any medium, provided you give appro-priate credit to the original author(s) and the source, provide a link to theCreative Commons license, and indicate if changes were made.

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